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Sphingolipids Containing Very-Long-Chain Fatty Acids Define
a Secretory Pathway for Specific Polar Plasma Membrane
Protein Targeting in Arabidopsis
W
Jonathan E. Markham,a,1,2 Diana Molino,b,1 Lionel Gissot,b Yannick Bellec,b Kian Hématy,b Jessica Marion,c
Katia Belcram,b Jean-Christophe Palauqui,b Béatrice Satiat-JeuneMaı̂tre,c and Jean-Denis Faureb,3
a Donald
Danforth Plant Science Center, St. Louis, Missouri 63132
Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique–AgroParisTech,
Centre de Versailles-Grignon, 78000 Versailles, France
c Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette Cedex, France
b Institut
Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional
analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for
the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant
development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of
lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1
in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–
labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane
structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a
trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.
INTRODUCTION
Sphingolipids are essential for eukaryotic life (Holthuis et al.,
2001). Current theory suggests this is due, at least in part, to their
role in protein sorting and secretion. Evidence indicates that,
within the diverse membrane composition of the Golgi body,
sphingolipids coalesce into microdomains or lipid rafts where,
together with cholesterol and saturated phospholipids, they
attract a unique subset of proteins and together are transported
to the plasma membrane (PM; Klemm et al., 2009). In animal
epithelial cells, this property of sphingolipids is exploited to
maintain cell polarity through the regulation of vesicle trafficking
and endocytosis at the apical membrane (Maier and Hoekstra,
2003; Nyasae et al., 2003). Sphingolipid sterol–rich microdomains are similarly recruited in the budding yeast Saccharomyces cerevisiae to establish cell polarity during mating and
budding (Bagnat and Simons, 2002).
The ability of sphingolipids to form microdomains may be
attributed to their unique physical properties compared with the
glycerolipids. Sphingolipids consist of three primary components:
an acyl amino alcohol or long-chain base (LCB), a fatty acid
1 These
authors contributed equally to this work.
address: Department of Biochemistry, University of Nebraska,
N146 Beadle Center, Lincoln, NE 68588.
3 Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Jean-Denis Faure
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.110.080473
2 Current
attached via the amino group, and a head group attached to
carbon-1 (C1) of the LCB. Additional hydroxyl groups at C2 on the
fatty acid and C4 on the LCB promote hydrogen bonding between
sphingolipids that is not available to glycerolipids (Pascher, 1976).
Furthermore, the fatty acid component of sphingolipids often
consists of a saturated or monounsaturated very-long-chain fatty
acid (VLCFA) of >18 carbons and up to 26 carbons in length (C26).
The presence of VLCFA in sphingolipids increases their hydrophobicity, membrane leaflet interdigitation, and the transition from
a fluid to a gel phase, which is a requirement for microdomain
formation. This important property of VLCFA in membrane organization is supported by the observation that S. cerevisiae
mutants unable to synthesize sphingolipids can be rescued by
the SLC1-1 mutation, which allows for the transfer of C26 fatty
acids to the sn-2 position of glycerolipids. C22 fatty acids are
unable to support the functions performed by C26 fatty acids,
indicating the sensitivity of the system to fatty acid chain length
(Gaigg et al., 2005, 2006). As a result, yeast mutants in which
fatty acid elongation beyond C16 is abolished are not viable (Oh
et al., 1997).
The incorporation of fatty acids into sphingolipids is catalyzed by
the enzyme ceramide synthase (sphinganine N-acyltransferase).
Ceramide synthases are encoded by the LAG1 gene family (named
after longevity assurance gene 1); members of which have been
found in all eukaryotes so far examined from fungi to animals and
plants (Winter and Ponting, 2002). In animals, several ceramide
synthases have been characterized (CERS1-6) and shown to have
different substrate specificities with respect to the length of the acyl
chain of the fatty acid (Riebeling et al., 2003; Mizutani et al., 2005,
2006). S. cerevisae contains two LAG1 family members, LAG1 and
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LAC1, and deletion of both of these genes causes strongly reduced
cell growth, cell wall defects, and delayed endoplasmic reticulum
(ER)-to-Golgi transport of glycosylphosphatidylinositol-anchored
proteins (Barz and Walter, 1999; Guillas et al., 2001; Schorling et al.,
2001). These phenotypes can be complemented by homologous
genes from Homo sapiens, Caenorhabditis elegans, and Solanum
lycopersicum (Jiang et al., 1998; Spassieva et al., 2002), indicating
that LAG1 homologs serve as ceramide synthases. Recent studies
on the role of the mammalian ceramide synthase CERS2 indicate
that it is responsible for the incorporation of the majority of VLCFAs
into the sphingolipids of the liver and brain (Imgrund et al., 2009;
Pewzner-Jung et al., 2010). Interestingly, the alteration in sphingolipid profile resulting from CERS2 disruption bears some resemblance to that obtained when challenged by the ceramide synthase
inhibitor fumonisin B1 (FB1), suggesting that FB1 may specifically
inhibit the incorporation of VLCFA into sphingolipids, thereby
mimicking the disruption of CERS2 activity (Pewzner-Jung et al.,
2010).
Recent studies have identified several mutants of acyl-CoA
elongation in plants that show phenotypes that have been
attributed to depletion of VLCFA in sphingolipids. These mutants
include cer10 (Zheng et al., 2005), gurke/pasticcino3 (pas3; Baud
et al., 2004), pas2 (Bach et al., 2008), and pas1 (Roudier et al.,
2010). In all these mutants, the level of VLCFA in sphingolipids is
reduced, and this reduction is coupled with important morphological changes in the plant. In the cer10 mutant, which is deficient
in elongation-specific enoyl reductase, endosomal compartments
were shown to accumulate, indicating abnormal vesicle trafficking
(Zheng et al., 2005). In the case of the pas1 mutant, abnormal
trafficking of the auxin polar efflux carrier PIN1 was associated
with decreased VLCFA in sphingolipids (Roudier et al., 2010). PIN1
is one of several proteins with a polar localization within the root
tissues of Arabidopsis thaliana responsible for forming auxin
gradients that in turn control root elongation and lateral root
formation. While VLCFAs are involved in other metabolic processes in plants, such as wax biosynthesis or seed storage lipid
biosynthesis, defects in neither of these lead to the extreme
defects in plant and seed morphology in these elongase mutants
leaving sphingolipids as the prime candidate.
Several lines of evidence suggest that sphingolipid sterol–rich
microdomains exist in plant membranes and that they may be
involved in the polar targeting of proteins to the PM (Fischer et al.,
2004). However, the precise role of sphingolipids in such processes has only been tangentially addressed in plants. As in
animals, plant sphingolipids are a complex family of lipids with
various acyl chain lengths, LCBs, and head groups moieties,
although the specific role of each moiety is still uncertain.
Complete structural analysis of sphingolipids has been carried
out in several plant species, identifying four main classes:
ceramide, hydroxyceramides, glucosylceramides, and glucosylinositolphosphorylceramides (Markham et al., 2006). Although
several steps of plant sphingolipid biosynthesis have been
identified and modified genetically, their effects on protein trafficking have not been adequately assessed. Recently, however,
inhibition of glucosylceramide synthase activity was shown to
correlate with protein trafficking in tobacco (Nicotiana tabacum),
suggesting that alteration in plant sphingolipid synthesis can
result in abnormal protein sorting (Melser et al., 2010).
The goal of this study was to identify the genes responsible for
ceramide synthase activity in Arabidopsis and to examine the
influence of sphingolipid content on the biology of a multicellular
organism. We discovered that the three Arabidopsis LAG1
homologs have different acyl chain substrate specificities and
that VLCFA sphingolipids are essential for plant development. By
combining genetic and pharmacological approaches, we show
that VLCFA-containing sphingolipids are required for organogenesis and, more specifically, for auxin-dependent lateral root
outgrowth. Finally, we demonstrate that the depletion of ceramides led to defective secretory targeting of AUX1 and PIN1 to
the PM by altering the Rab-A2a early endosomal pathway,
suggesting that VLCFA sphingolipids define specific endomembrane compartments for targeting specific polar cargo proteins.
RESULTS
Identification of Genes for Ceramide Synthase
in Arabidopsis
The first ceramide synthase identified from plants is the tomato
gene Asc-1 (for Alternaria stem canker resistance-1) that gives
resistance to the ceramide synthase inhibitor FB1 (Brandwagt
et al., 2000; Spassieva et al., 2002). A tBLASTn search of the
completed Arabidopsis genome identified three Asc-1/LAG1 homologs in Arabidopsis, At3g25540, At3g19260, and At1g13580,
subsequently referred to as LOH1, LOH2, and LOH3, respectively
(LAG One Homologue). A potential fourth homolog (At1g26200)
was also identified; however, it contains some highly variable sequences not found in any other LAG1 homologs that may suggest
a different function yet to be discerned (see Supplemental Figure
1A online). The protein sequences of the identified LAG1 homologs
were aligned with the known full-length proteins from tomato and
the LAG1 and LAC1 proteins from yeast to identify conserved
structural features (Figure 1A). The alignments show strong conservation in the LAG1p motif and predicted transmembrane
helices, suggesting that the LOH genes encode ceramide synthases. A phylogenetic tree derived from an alignment of all known
full-length protein sequences of the eudicot plant LAG1 homologs
along with the LAG1/LAC1 proteins from yeast and the CerS
proteins from human was constructed (see Supplemental Figures
1A and 1B and Supplemental Data Set 1 online). The tree showed
that LOH1 and LOH3 are closely related and cluster into a group
along with the majority of other plant LAG1 homologs (see
Supplemental Figure 1B online). On the other hand, LOH2 appears
to be evolutionarily distinct from LOH1 and LOH3. In order to
investigate the function of the LOH genes, genetic disruptions
were characterized for each gene.
Arabidopsis LAG1 Homologs LOH1 and LOH3 Are Essential
for Plant Growth
T-DNA insertion mutants were identified from the Wisconsin
(Krysan et al., 1999) (loh1-1, loh2-1, and loh3-1) and Salk (Alonso
et al., 2003) (loh1-2, loh2-2, and loh3-2) collections for each of
the three Arabidopsis LOH genes (Figure 1B). Both alleles loh1-1
and loh3-1 are insertions in the 59-untranslated region of their
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Figure 1. LOH1 and LOH3 Are Essential for Plant Development.
(A) Alignment of ceramide synthase protein sequences from Arabidopsis (LOH), tomato (Le ASC), and S.s cerevisae (Lag1p and Lac1p). Boxes indicate
predicted transmembrane domains; an extratransmembrane domain, TM0, is predicted in S. cerevisae. Black shading shows the residues completely
conserved among the ceramide synthase family, and gray shading indicates partial conservation (dark and light gray for seven and four to six residues,
respectively, conserved out of eight).
(B) Structure of the Arabidopsis LOH genes and alleles. Boxes represent introns, with dark blue representing the coding region. The positions of
identified T-DNA insertions in the different alleles are marked by triangles.
(C) Seeds and embryos from the wild type (left) and loh1-2 loh3-2 double mutants (right) showing shrunken seeds and abnormal embryo development.
Bars = 100 mm
(D) Germinating loh1-2 loh3-2 seedlings showing very slow growth at 2 weeks (left) and no root development and abnormal leaf shape at 4 weeks (right).
Bars = 2 mm.
(E) Phenotype of loh1-1 loh3-1 mutants compared with the wild type (top left). Mutants show developmental defects ranging from modification of
cotyledon number (top right) or shape (top middle and bottom left) to complete arrest of growth (bottom middle) and even severely impaired embryo
morphology (bottom right). Bars = 300 mm.
(F) Adult loh1-1 loh3-1 mutants showed reduced size with smaller rosette and shorter stems. Bar = 3 cm.
respective genes (at 23 and 210 bp from the ATG start, respectively) leading to knockdown of wild-type mRNA levels (see
Supplemental Figure 2A online). All other mutants are caused by
insertions in an exon of their respective genes and result in
undetectable wild-type mRNAs and are therefore true knockouts
(see Supplemental Figures 1A and 1B online).
Despite this, each single mutant showed a normal phenotype
under standard growth conditions (see Supplemental Figure 2C
online). Similarly, combinations of double mutants between loh2
and loh1 or loh3 null alleles did not show any significant differences of growth or development (see Supplemental Figure 2C
online). By contrast, the cross between the knockout alleles
loh1-2 and loh3-2 resulted in ;1:16 seeds that were darker
and smaller with a poor germination rate in the F2 progeny (n =
300). This phenotype was confirmed in the F3 progeny of the
sesqui-mutant combinations loh1-22/2 loh3-2+/2 (n = 96) or
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loh1-2+/2 loh3-22/2 (n = 79), which produced 18 and 12%
abnormal seeds, respectively (Figure 1C). The embryo morphology in these seeds was often strongly altered with thicker hypocotyls and a single cotyledon (Figure 1C). No double loh1-2 loh3-2
mutant was recovered from seeds sown on soil, suggesting that
the combination of mutations is lethal. However, when sown on
solid media containing Suc, seedlings of the loh1-22/2 loh3-22/2
genotype could sometimes be recovered, albeit after a prolong
incubation of several weeks. The resulting seedlings showed severely deformed plants with no roots and a deformed leaf shape
that did not further develop, indicating that LOH1 and LOH3 are
essential for root growth (Figure 1D). Despite transfer to fresh media,
none of the seedlings were able to survive longer than 4 weeks.
As the loh1-2 loh3-2 double mutant is lethal, the cross between
the knockdown alleles loh1-1 and loh3-1 was also examined.
Like the loh1-2 loh3-2 seeds, loh1-1 loh3-1 seeds showed reduced viability, and those that did germinate gave rise to
seedlings with highly variable phenotype ranging from triple
cotyledon, single deformed cotyledon, or undeveloped cotyledon (Figure 1E). Plants with relatively normal development
showed smaller, wrinkled leaves, early senescence, delayed
flowering, and reduced primary bolt length (Figure 1F; see
Supplemental Figures 2G and 3D online). Embryos dissected
from seeds that did not germinate showed very strong alteration
of cotyledon morphology with often single cotyledon embryos
(Figure 1E) similar to the phenotypes observed in the progeny of
sesqui-mutant combinations. To confirm that these phenotypes
were indeed caused by the loss of LOH1 and LOH3 function,
native LOH1 and LOH3 genes were transformed into the loh1-1
loh3-1 or sesqui loh1-2 3-2+/2 mutants under the control of the
native promoter (see Supplemental Figures 3A and 3E online).
Plants with a wild-type phenotype were recovered upon transformation with either LOH1 or LOH3 (see Supplemental Figures 3B to
3D online), indicating that the mutations in the LOH1 and LOH3
genes are responsible for the phenotypes observed in each of the
double mutants. Overall, these data demonstrate that, out of the
three different LOH genes, LOH1 and LOH3 are redundant and
essential for plant growth, while LOH2 has no obvious effect on
plant development, indicative of a different role for LOH2 versus
LOH1 and LOH3 in sphingolipid metabolism.
LOH Genes Encode Ceramide Synthases with Different
Specific Activities
To investigate the role of the different LOH genes in sphingolipid
metabolism and the biochemical basis for the phenotypes of
the loh mutants, a complete sphingolipid analysis was performed
for each line (Markham and Jaworski, 2007). For the lethal
loh1-2 loh3-2 double mutant, sphingolipid analysis performed on
2-week-old germinated seeds grown on solid media showed that
all the classes of sphingolipids from these mutants were almost
completely devoid of VLCFA (Figure 2A; see Supplemental
Figure 4 online) and instead contained mostly 16:0 fatty acids.
To confirm these results, sphingolipid analysis was performed on
seedlings from the knockdown loh1-1 loh3-1 double mutant. As
expected, this mutant combination showed a less drastic reduction in VLCFA containing sphingolipids, ;30 mol % in total
(Figure 2B; see Supplemental Figure 4A online). Single loh1-2 or
loh3-2 mutants had sphingolipid profiles almost identical to the
wild type, indicating that LOH1 and LOH3 have redundant
activities specifically targeted toward VLCFA sphingolipids (see
Supplemental Figure 4A online). The combined loss of LOH1 and
LOH3 was also associated with a very strong accumulation of
saturated free LCBs consistent with reduced ceramide synthase
activity in these mutants (Figure 2C). Free LCBs also accumulated in the loh1-1 loh3-1 mutants, albeit less pronounced than
for loh1-2 loh3-2 (see Supplemental Figure 4B online).
Because disruption of LOH1 and LOH3 leads to an almost
complete absence of VLCFA in sphingolipids, this suggests that
the remaining ceramide synthase in these plants, LOH2, must
be specific for 16:0. In support of this, sphingolipid profiling of
loh2-1 and loh2-2 seedlings showed a strong reduction of 16:0
fatty acid in ceramide and other sphingolipids and a significant
increase in VLCFA containing sphingolipids (Figure 2A; see Supplemental Figure 4A online). Altogether, these results demonstrate
the existence of at least two different ceramide synthase activities
in Arabidopsis: LOH1 and LOH3 are specific for VLCFA, while
LOH2 is specific for 16:0 fatty acids. Interestingly, disruption of
LOH1 and LOH3 leads to high accumulation of free LCBs, similar
to what was seen when CERS2 is disrupted in mice (PewznerJung et al., 2010) and reminiscent of the effect of ceramide
synthase inhibitors such as FB1 and AAL toxin (Abbas et al., 1994).
FB1 Mimics the Disruption of VLCFA-Specific
Ceramide Synthases
FB1 and AAL toxins are sphingoid-base analog mycotoxins that
have a strong and specific inhibitory effect on ceramide synthases (Merrill et al., 1993; Spassieva et al., 2002). Previous
studies in animal systems have suggested that FB1 may be
selective in its inhibition of ceramide synthase activity because
treatment with FB1 changes the profile of fatty acids incorporated into sphingolipids (Venkataraman et al., 2002). The effect of
0.5 mM FB1 on the sphingolipid profile of Arabidopsis seedlings
was examined (Figure 2D). Counterintuitively, after 9 d of growth
on FB1, Arabidopsis seedlings contained higher amount of total
sphingolipids than control plants, but the majority of the extra
sphingolipids consisted of 16:0 species with a decrease in VLCFA
containing species (Figure 2D). Exposure to FB1 for 24 h or
less did not cause excessive 16:0-ceramide to be synthesized;
however, a small, but significant, reduction in VLCFA-containing
ceramide was noticeable by 16 to 24 h (see Supplemental Figure
5A online). Inhibition of ceramide synthase by FB1 was detectable
even after short exposure to FB1, judging by the rapid accumulation of the free trihydroxy (t) LCBs t18:0 after 4 h of treatment (see
Supplemental Figures 5B and 5C online). Accumulation of t18:0
and the dihydroxy (d) d18:0 was directly correlated with the time of
exposure to FB1 with a maximum at 8 and 16 h for t18:0 and d18:0,
respectively. Application of FB1 for 16 h and longer was saturating
as shown by free LCB levels (see Supplemental Figure 5B online).
The effect of FB1 was also dose dependent, as shown by the analysis of total LCB hydrolyzed from sphingolipids after 9 d exposure to 0.4 and 0.8 mM FB1 (see Supplemental Figure 5D online).
Together, these data indicate that FB1 exposure rapidly inhibits
ceramide synthase, elevating free LCB levels and depleting
VLCFA-containing ceramides. Eventually, free LCBs are rerouted
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Figure 2. Disruption of LOH Activity by Knockout or FB1 Treatment Alters Sphingolipid Content.
(A) Total sphingolipid content of loh2-2 and loh1-2 loh3-2 compared with their related control (Col-0). Sphingolipid content is shown according to the
four different classes (ceramides, hydroxyceramides, glucosylceramides, and glucosylinositolphosphorylceramides) and the length and saturation of
the fatty acid chain.
(B) Total sphingolipid content of loh1-1 loh3-1 compared with their related control (WS). Sphingolipid content is shown as in (A).
(C) Free LCB levels of loh2-2, loh1-2 loh3-2, and wild-type seedlings grown in the presence of 0.5 mM FB1 for 9 d compared with the Col-0 wild type.
(D) Total sphingolipid content of wild-type (Col-0) seedlings grown in presence or absence of 0.5 mM FB1 for 9 d was compared with the loh1-1 loh3-1
mutant and its corresponding wild-type seedlings (Ws). Sphingolipid levels are shown as in (A).
LCB(P)s are referred to using standard sphingolipid annotation as explained by Markham and Jaworski (2007). Measurements are the average of three
to five replicates.
into 16:0-ceramides similar to the disruption of sphingolipid metabolism seen in the loh1 loh3 double mutants. As expected,
knockdown loh1-1 loh3-1 double mutants treated with FB1
showed stronger depletion of VLCFA sphingolipids and enhanced
accumulation of 16:0 sphingolipids, confirming the specific targeting of LOH1 and LOH3 ceramide synthases by FB1 (see Supplemental Figure 5E online).
Wild-type seedlings germinated for 9 d on FB1 concentrations of
1 mM or higher showed global inhibition of growth (Figure 3A). At 0.5
mM FB1, primary root length was only moderately reduced (30%
reduction compared with untreated control), but a strong reduction
of lateral root emergence could be observed on 1 mM FB1 with
80% reduction compared with untreated control (Figure 3B).
Likewise, knockdown loh1-1 loh3-1 mutants, displaying abnormal
cotyledon symmetry, also showed a 60% reduction in lateral root
numbers compared with wild-type controls, as did the two different
combinations of sesqui-mutants involving the loh1-2 loh3-2 alleles
(loh1-22/2 loh3-2-/+ or loh1-2 -/+ loh3-22/2) (Figure 3C; see Supplemental Figure 2D online). The weakest mutant phenotype could
be mimicked by decreasing FB1 concentration to 0.25 mM, leading
to 50% inhibition of lateral root growth (Figure 3B). FB1 application
reduced lateral root primordia outgrowth but not the initiation
stages as detected by the auxin reporter DR5:b-glucuronidase
(GUS) (see Supplemental Figure 2E online). Lateral root formation
and growth is known to be very dependent on auxin, suggesting
that the strong morphological changes observed in the loh1/loh3
mutants could be related to an altered auxin physiology. Interestingly, the pas1 mutant has reduced VLCFAs in sphingolipids and
showed auxin-related inhibition of lateral root formation (Roudier
et al., 2010). Primary root growth of pas1 was found to be more
sensitive to FB1 compared with the wild type, supporting the
involvement of VLCFA sphingolipids in auxin-dependent lateral
root formation (see Supplemental Figure 6A online).
Very-Long-Chain Ceramides Are Required for Polar
Auxin Distribution
In order to analyze the effect of sphingolipid composition on
auxin distribution, we analyzed DR5:GUS expression in roots
grown in presence of FB1 (Swarup et al., 2008). DR5:GUS
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Figure 3. Inhibition of Ceramide Synthase Leads to Altered Root Development and Auxin Transport.
(A) and (B) Primary (A) or lateral (B) root length of Arabidopsis seedlings grown in the presence of various concentrations (0, 0.5, 1, and 3.5mM) of FB1.
Shown is the average of 17 to 35 measurements plus standard error. Bars = 3 mm in (A) and 1 mm in (B).
(C) Measurement of primary root growth and lateral root development in wild-type (Ws) and loh1-1 loh3-1 mutant plants of 9 d. Number of lateral roots is
the average of 16 to 35 plants normalized against wild-type plus standard error. Bar = 3 mm.
(D) DR5:GUS staining in 7-d-old wild-type primary root meristems treated (+) or not treated ( ) with 2.5 mM FB1 for 16 h. Bar = 100 mm.
(E) and (F) Wild-type (Ws), FB1-treated, or loh1-1 loh3-1 mutant plants grown in the absence ( ) or presence (+) of the auxin analog NAA. The effect of
exogenous NAA treatment on lateral root development in the presence of FB1 was quantified by counting the lateral roots in 18 to 23 plants and
normalizing against the wild type (F). Shown are the mean plus standard error. Bars in (E) = 3 mm.
(G) Transport of exogenous auxin through stem segments of wild-type plants (Ws, gray bars) and loh1-1 loh3-1 mutant plants (white bars). Transport
was measured in segments taken from different portions of the inflorescence stem (bottom, middle, and top) and in a basipetal or acropetal direction
and in the presence (+NPA) or absence of NPA. Significant (P # 0.05; Student’s t test) differences from control plants are indicated by an asterisk.
expression was reduced in root tips of seedlings grown for 9 d in
the presence of 1 mM FB1 (see Supplemental Figure 2F online) or
simply treated with 2.5 mM FB1 for 16 h (Figure 3D). To check if
auxin was directly involved in the root response to inhibition of
ceramide synthase, we tested the effect of the permeable auxin
analog naphthalene acetic acid (NAA) on lateral root development
of FB1-treated seedlings and loh1-1 loh3-1 double mutants. In
both cases, NAA was able to restore lateral root outgrowth,
suggesting that auxin transport and not auxin response was most
likely impaired in FB1-treated roots (Figures 3E and 3F).
To investigate the role of VLCFA-containing sphingolipids in
auxin transport, the transport of radiolabeled auxin along stem
segments of wild-type and loh1-1 loh3-1 mutants was measured. Stem segments of 2.5 cm in length were incubated for 16 h
in the dark with the top/flower end (basipetal) or bottom/rosette
end (acropetal) in a 20-mL solution of 14C-labeled indole-3-acetic
acid, the native auxin. The basipetal or acropetal auxin movements were subsequently quantified by measuring the amount of
labeled auxin in the distal 5 mm along the stem. Controls for
ruling out possible diffusion or metabolism of indole-3-acetic
acid were performed by including 10 mM naphthylphalamic acid
(NPA), an inhibitor of polar auxin transport. As expected, labeled
auxin showed significant, NPA-sensitive, basipetal transport
along the different sections of the wild-type stem (Figure 3G).
Sphingolipids and Polar Auxin Transport
In the loh1-1 loh3-1 mutant, the auxin flux was strongly affected
compared with the wild type with the amount of auxin transported reduced by 50 to 75% along the stem (Figure 3G).
Together, these data show that polar auxin transport is impaired
by the reduction in VLCFA in the loh1-1 loh3-1 mutant or by the
inhibition of ceramide synthase in the FB1-treated plants.
Sphingolipid Synthesis Is Required for the Targeting of
Specific Auxin Carriers to the PM
Polar auxin transport is mediated through a family of PM polar
auxin transporters involved in cellular influx (AUX1 family members) or efflux of auxin (PIN family members). Polar localization of
auxin transporters was thus investigated in the context of sphingolipid depletion either in the loh1 loh3 mutant or in FB1-treated
seedlings. To minimize possible artifactual response at the
cellular level induced by long FB1 treatment, we designed a
shorter FB1 treatment leading to ceramide synthase inhibition.
We found that the application of 2.5 mM FB1 for 16 h (subsequently referred as FB1-16h) led to an accumulation of t18:0 and
d18:0 that was identical to that induced by 9 d of treatment with
0.5 mM (see Supplemental Figure 5B online). Since FB1 was
described to induce cell death at high concentration, we verified
that our short FB1 treatment was not impairing cell viability. We
monitored cell viability in both primary and lateral roots using the
fluorodiacetate (FDA) test, which labels viable cells by monitoring
fluorescein release from ester bond hydrolysis of apolar FDA. In
our conditions, FB1-treated roots were still showing fluorescein
labeling, demonstrating their viability (see Supplemental Figure
6B online). This experimental setting allowed the study of cellular
responses in lateral root tip, which was found to be more
sensitive to ceramide synthase inhibition than the primary root
(Figures 3A and 3B).
We first analyzed AUX1-yellow fluorescent protein (YFP) distribution since aux1 mutants showed reduced lateral root development similar to FB1-treated plants (Marchant et al., 2002). FB1
treatment for 16 h led to an accumulation of pAUX1:AUX1-YFP
fluorescence inside meristematic, protophloem, and epidermal
cells of lateral roots (Figures 4A and 4B). Similar aggregation of
AUX1-YFP was observed in the primary root of sesqui loh1-2+/2
loh3-22/2 mutants but not in the weak loh1-1 loh1-3 (Figure 4C).
PM targeting of AUX1-YFP was also impaired in lateral root
primordia of loh1-2+/2 loh3-22/2 (see Supplemental Figure 7A
online). Interestingly, FB1 treatment led to partial relocalization of
AUX1-YFP into lateral membrane of epidermal and protophloem
cells (Figures 4A and 4C, inset, arrows). We also investigated
whether inhibition of ceramide synthase would have a similar effect
on the subcellular distribution of LAX3-green fluorescent protein
(GFP), since this AUX1-like influx carrier was described to be also
essential for lateral root outgrowth (Swarup et al., 2008). But
contrary to AUX1-YFP, FB1 did not modify LAX3-GFP targeting to
the PM (see Supplemental Figures 7B and 7C online).
We then checked the effect of FB1-16h treatment on the
subcellular distribution of auxin efflux carriers in protophloem
and epidermal cells. Like AUX1-YFP, PIN1-GFP was found to
aggregate both in vascular and root meristem cells of lateral
roots in presence of FB1 or in the most affected primary root of
the weak loh1-1 loh3-1 mutant (Figure 4D; see Supplemental
7 of 17
Figures 8A and 8B online). In the most severe cases, PIN1-GFP
or PIN1 immunolocalization labeling showed PIN1 almost exclusively in cytosolic aggregates and no or only a weak signal could
be detected at the PM (Figure 4D; see Supplemental Figure 8C
online). However, subcellular distributions of other efflux carriers,
such as the ABC1-GFP and ABC19-GFP auxin pumps, were not
affected by FB1-16h treatment (Figures 4E and 4F). The auxin
efflux carrier PIN2-GFP was also insensitive to FB1 (see Supplemental Figure 7D online); however, this was unrelated to its
epidermal localization because PIN1-GFP expression in the
epidermis led to aggregates as in the stele (see Supplemental
Figure 7E online). We also checked PM proteins not involved in
auxin transport, such as the nonpolar LTi6b-GFP and PIP2-GFP
fusion proteins. As for ABCs, PIN2, or LAX3 proteins, the localization
of these markers was insensitive to FB1 treatment (see Supplemental Figures 7F and 7G online). Altogether, these results indicate
that ceramide synthase inhibition modified membrane targeting of
specific protein cargoes. Interestingly, even if AUX1 and PIN1 are
targeted to different sites within the PM, both proteins gathered
partially into the same or closely associate aggregates upon FB1
application (Figure 4G; see Supplemental Figure 8C online).
Inactivation of LOH activity led not only to decreased VLCFAcontaining ceramide and sphingolipid contents but also to the
accumulation of free LCBs. To exclude a possible role of free
LCBs in the aggregation of AUX1 and PIN1 proteins, we treated
Arabidopsis AUX1-YFP and PIN1-GFP seedlings with myriocin,
an inhibitor of serine palmitoyltransferase, the first step of sphingolipid synthesis, leading to the depletion of both free LCBs and
ceramides (Miyake et al., 1995). Like FB1, myriocin treatment led
to similar accumulation of AUX1-YFP in cytosolic aggregates,
demonstrating that inhibition of de novo sphingolipid synthesis
and not LCB accumulation was responsible for the specific
mistargeting of protein cargoes (see Supplemental Figure 8D
online). In conclusion, our results showed that inhibition of
sphingolipid synthesis impaired normal trafficking of AUX1-YFP
and PIN1-GFP proteins in the protophloem, meristem, and
epidermal root cells, explaining the reduction of polar auxin
transport and auxin-dependent lateral root outgrowth.
Inhibition of Sphingolipid Synthesis Disrupts
Early Endosomes
Inhibition of ceramide synthase induced the aggregation of
membrane and proteins in a structure that could be referred to
as FB1 compartment by analogy with compartments induced by
the anterograde transport inhibitor brefeldin A (BFA). We therefore investigated the different endomembrane markers recruited
in the formation of these compartments. GFP marker lines for
different endomembrane compartments were tested for their
sensitivity to FB1. First, we investigated the effect of FB1 on the
ER, since it is the site of de novo LCB, ceramide, and probably
glucosylceramide biosynthesis (Marion et al., 2008). We examined the distribution of the ER markers YFP-NIP1 and AXR4GFP. AXR4-GFP was of special interest since axr4 mutant
aggregated AUX1 into cytosolic compartments, blocking its
targeting to the PM (Dharmasiri et al., 2006). No obvious ER
containing aggregates could be observed upon FB1-16h treatment for both markers, although enhanced AXR4-GFP signal
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was often observed in the perinuclear region (Figure 5A). We then
evaluated the localization of several markers labeling the ER/
cis-Golgi and Golgi apparatus, but none of them showed any
significant aggregation in presence of FB1 (Figure 5). By contrast, the early endosomal markers YFP-Rab-A2a and YFPRabA1e were clearly aggregated by FB1-16h treatment (Figure
5). The RabA2a compartment extensively overlapped with the
trans-Golgi network (TGN)-resident VHAa1 (Chow et al., 2008).
As expected, partial colocalization of YFP-RabA2a and VHAa1RFP could also be observed in FB1-induced aggregates (Figure
5B). Endosomal subpopulations labeled by YFP-ARA7/RabF2b
or SNX1-GFP, defined as late endosomes, were insensitive to
FB1 application. Similarly, late endosome vacuolar YFP-RabG3C
or YFP-RabG3F or specific vacuolar marker YFP-VAMP711 were
also insensitive to FB1 (Figure 5A; see Supplemental Figure 8E
online). Altogether, these results indicated that inhibition of
ceramide synthase modified subcellular distribution only of the
early endosomal/TGN resident markers, suggesting the involvement of sphingolipid synthesis in the maintenance of specific
endomembrane compartments.
The modifications of endomembrane structures observed with
GFP markers were confirmed by transmission electron microscopy. The organization of the Golgi apparatus as well as the
mitochondria and plastids were not modified by FB1-16h treatment and by loh1-1 loh3-1 mutations (Figure 6; see Supplemental Figures 9A and 9B online). The ER network was clearly visible,
and it seemed to be larger and more branched, an observation
that could correlate with higher fluorescence of ER markers after
FB1 treatment. The most dramatic changes were the presence of
small vesicular structures looking like dismantled vacuole or
fused vesicles (Figure 6D). Since fluorescent vacuolar markers
did not show major macroscopic changes in vacuolar structure
while early endosomal markers formed large aggregates, these
accumulations of membrane materials could result from fusion of
endosomal compartments.
Sphingolipid Synthesis Is Not Required for Endocytosis
AUX1-YFP and PIN1-GFP aggregates in the cytosol most probably result from altered TGN and/or early endosomal trafficking.
However, PIN1 and AUX1, apart from being localized in the
opposite side of the cell, are characterized by different dynamics with PIN1 undergoing active recycling, while AUX1
showed a slower turnover at the PM (Kleine-Vehn et al., 2006).
Figure 4. Inhibition of Ceramide Synthesis Alters the AUX1-YFP and
PIN1-GFP Targeting in Lateral Roots.
(A) and (B) AUX1-YFP aggregates (arrowheads) in the cytosol in presence
of 2.5 mM FB1 for 16 h (A). Detail of untreated (left, ) and treated lateral
root cells (right, +). Details of epidermal cells are given in insets. Details of
protophloem region of untreated and treated lateral roots are shown in (B).
(C) AUX1-YFP aggregates (arrowheads) in the cytosol of meristematic and protophloem cells in the primary root of sesqui-mutants
loh1-2 / loh3-2 +/ (right) compared with the wild type (wt; left).
(D) PIN1-GFP aggregates (arrowheads) in the cytosol of FB1-treated
cells (bottom, +) compared with control (top, ). PIN1 aggregates were
also confirmed by immunolocalization (anti-PIN1). Note that FB1 treatment depleted PIN1 from PM.
(E) ABC1-GFP expression in untreated (left, ) and FB1-treated cells
(right, +).
(F) ABC19-GFP expression in untreated (left, ) and FB1-treated cells
(right, +).
(G) AUX1-YFP (red) and PIN1-GFP (green) distribution in absence (left,
FB1) and in presence of FB1 (right, +FB1). Merged images are also
shown. AUX1-YFP and PIN1-GFP aggregates colocalize partially (arrowheads).
Bars = 20 mm in (A), (F), and (G) and 10 mm in inset of (A) to (E) and (H).
Sphingolipids and Polar Auxin Transport
9 of 17
Figure 5. Inhibition of Ceramide Synthase Affects Specific Endomembrane Compartments.
(A) Fluorescence micrographs showing the subcellular localization of different endomembrane marker-XFP fusions in the lateral roots of untreated
transgenic lines ( FB1) or after treatment with FB1 (+FB1). The subcellular compartment to which the marker has previously been localized is annotated
far right or left, and the marker-XFP fusion being examined is shown in the top left of each set of micrographs. Note the aggregations under FB1
treatment for the early endosome markers (arrowheads). Bars = 5 mm.
(B) TGN marker VHAa1-RFP partially colocalizes with YFP-RabA2a in the presence of FB1. Bars = 5 mm.
We thus reasoned that the aggregation of both proteins upon
ceramide synthesis inhibition would preferentially result from
secretory pathway impairment rather than PM recycling. To first
evaluate the involvement of recycling membrane material in
ceramide-depleted endomembrane pools, we checked whether
endocytosed membrane material was associated with FB1
compartments. Early endosomes like Rab-A2a rapidly colocalized with endocytosed FM4-64 (Chow et al., 2008). However,
when treated with FB1, the aggregated YFP-Rab-A2a–labeled
endosomes showed close proximity with FM4-64 but did not
seem to colocalize, indicating that endocytosis provides only a
minor contribution to the formation of FB1 compartments (see
Supplemental Figure 10A online). This was confirmed by comparing BOR1-GFP internalization upon boron application in
presence or not of FB1-16h treatment (Takano et al., 2005).
BOR1-GFP–labeled endosomes appeared with similar kinetics
in the presence or absence of FB1, indicating that endocytosis
was not severely impaired by FB1-16h treatment (see Supplemental Figure 10B online). The origin of the FB1 compartment
was directly probed using point mutations in Rab-A2a that modify
its subcellular distribution (Chow et al., 2008). To rule out possible interaction of GTPase activity with FB1 treatment, we
monitored the subcellular distribution in a YFP-Rab-A2a[N125I]
mutant that is a nucleotide-free dominant-negative mutant that
has the same subcellular distribution as wild-type YFP-Rab-A2a
but lower expression levels. YFP-Rab-A2a[N125I] was as sensitive to FB1 as wild-type YFP-Rab-A2a, demonstrating that
FB1 sensitivity was independent of GTPase activity (see
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Figure 6. Inhibition of Ceramide Synthase Alters Endomembrane Ultrastructure.
(A) and (B) Control cells exhibiting characteristic tubular ER and a Golgi stack (GA) surrounded by secretory vesicles (A). Detail of control cells exhibiting
a narrow cell wall domain and a straight cell surface between two cells (B). NM, nuclear membrane.
(C) to (G) Cells from roots of seedlings grown on 0.5 mM FB1 for 9 d.
(C) Golgi ultrastructure is not affected by FB1 treatment.
(D) Cytoplasm is often filled with small, deconvoluted vacuole-like structures.
(E) and (F) The ER exhibits a larger and more branched network compared with control cells.
(G) The PM is detached from the cell wall, and a large and irregular periplasmic space between the cell wall and PM is observed.
Bars = 200 nm in (A), (C), (D), and (F), 500 nm in (B) and (G), and 1 mm in (C) and (E).
Supplemental Figure 10C online). Then, we analyzed the S26N or
Q71L mutations that stabilize YFP-Rab-A2a into the inactive
GDP-bound state or the constitutively active GTP-bound state.
These mutations relocate YFP-Rab-A2a mainly to the Golgi for
YFP-Rab-A2a[S26N] and the PM for YFP-Rab-A2a[Q71L]. The
Golgi YFP-RabA2a[S26N] was still sensitive to FB1 by forming
aggregates, while the PM-localized YFP-Rab-A2a[Q71L] was
insensitive, demonstrating that FB1 aggregates recruited YFPRab-A2a from the Golgi but not the PM (see Supplemental Figure
10C online). Altogether, these results indicate that FB1-16h
compartments are not formed from recycled PM materials.
Sphingolipid Synthesis Is Required for the AUX1 and PIN1
Secretory Pathway
The alternative pathway for FB1 compartments would be the
secretory route. BFA inhibits anterograde transport, resulting
also in the aggregation of early endosomes with the TGN to form
BFA compartments (Ritzenthaler et al., 2002; Geldner et al.,
2003). BFA was thus applied to FB1-treated PIN-GFP and AUX1YFP roots stained with FM4-64. In vascular cells, AUX1-YFP and
PIN1-GFP FB1-induced aggregates appeared to be distinct from
BFA compartments, suggesting that FB1 impaired a distinct
Sphingolipids and Polar Auxin Transport
11 of 17
post-Golgi compartment than did BFA (Figures 7A to 7D; see
Supplemental Figures 11A to 11D online). To address the involvement of de novo synthesis in FB1 aggregates, we directly
monitored aggregate formation through whole-cell fluorescence
recovery after photobleaching (FRAP) analysis. We performed
FRAP analysis of FB1-16h–treated PIN1-GFP and AUX1-YFP
secondary root cells. FB1-16h– treated cells with PIN1-GFP
localized in aggregates and at the PM were completely bleached
(Figure 7E). Recovery of PIN1-GFP in aggregates was clearly
visible after 30 min, and PM labeling was observed after aggregate recovery usually 30 to 45 min after bleaching. A similar
finding was observed with AUX1-YFP, albeit with a reduced
recovery (see Supplemental Figure 11E online). FRAP analysis
showed that FB1 delayed but also reduced PIN1-GFP recovery
at the PM (Figure 7E, left). However, PIN1-GFP recovery in
aggregates showed similar kinetics than the recovery of PIN1GFP at PM in untreated samples (Figure 7E, right), indicating that
the primary effect of FB1 is to block secretion by aggregating
PIN1-GFP prior to its PM localization. Altogether, these results
show that PIN1-GFP, like AUX1-YFP, aggregation occurs along
the secretory pathway.
Finally, we investigated the involvement of VLCFA sphingolipids in protein secretion in the apoplast by analyzing the
localization of tomato arabinogalactan fusion protein LeAGP1GFP (see Supplemental Figure 11F online). It was previously
demonstrated that this fusion protein provided a robust marker
for secretion in Arabidopsis (Estévez et al., 2006). FB1-16h
treatment did not modify LeAGP1 accumulation in the apoplast,
confirming that VLCFA sphingolipids are involved in the traffic of
specific cargoes along the secretory pathway.
DISCUSSION
Functional analysis of Arabidopsis LAG1 homologs 1 through 3
demonstrated that the three LOH genes encode ceramide synthases. The three LOH proteins showed high sequence identity
with the yeast LAG1p and LAC1p but also with tomato Le Asc1,
which was demonstrated to complement yeast lag mutants
(Spassieva et al., 2002). Sphingolipid analysis of the different loh
mutants demonstrated how the different LOH proteins contribute
to ceramide biosynthesis and more specifically, the sphingolipid
fatty acid chain length. Complete loss of LOH2 function led to
specific reduction in C16-ceramide, whereas the absence of
both LOH1 and LOH3 activity caused complete depletion of
ceramides with an acyl chain longer than C18. Altogether, these
results show that Arabidopsis possesses two different ceramide
synthase activities. LOH2 encodes long-acyl-chain ceramide
synthase, whereas LOH1 and LOH3 encode ceramide synthases
with very-long-acyl-chain specificity. The role of the distinct
LAG1 homolog At2g26200 in plant biology remains somewhat of
an enigma as LOH1, 2, and 3 would appear to account for VLCFA
and c16-specific ceramide synthase activity in leaf tisues. This
functional distinction is supported by the phylogenetic analysis
of plant LOH proteins, with LOH2 representative of a distinct
clade. It will be interesting to see if other members of the LOH2
clade are also specific for C16-fatty acids and to dissect the
amino acid differences between LOH2 and VLCFA-specific
Figure 7. Inhibition of Ceramide Synthase Modifies Vesicle Trafficking.
(A) Partial localization of pAUX1:AUX1-YFP and FM4-64 staining in the
presence of 50 mM BFA.
(B) Detail of BFA compartments with presence (open arrowhead) or
absence (closed arrowhead) of colocalization between FM4-64 and
AUX1-YFP.
(C) FM4-64 staining of aggregates in FB1-16h–treated pAUX1:AUX1YFP cells in the presence of BFA. Partial colocalization is observed
(arrowheads).
(D) Detail of FM4-64 staining of aggregates in FB1-treated pAUX1:AUX1YFP cells showing partial colocalization of AUX1-YFP with FM4-64 in
FB1/BFA compartments.
(E) FRAP analysis of FB1-16h cells expressing PIN1-GFP. Lateral root
cells expressing PIN1-GFP (prebleach) were bleached, and recovery of
PIN1-GFP was monitored for 60 min. Aggregates are shown (arrows).
Fluorescence recovery was quantified on transverse membranes in the
absence (PM ) or the presence of FB1 (PM +) as well on aggregates
(AG+) (n = 10, average + SD). Inset shows the two types of ROI used for
FRAP quantification.
Bars = 10 mm in (A), (C), and (E) and 5 mm in (B) and (D).
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The Plant Cell
ceramide synthases. Similar differences of acyl chain specificity
were also described for mammalian ceramide synthase CERS
(Teufel et al., 2009). CERS5 and 6, like LOH2, display a preference for long-chain acyl-CoA, while CERS2 or 4 is specifically
involved in the synthesis of C22- and C22-C24-ceramides,
respectively.
In yeast, both ceramide synthases are specific to very-longchain ceramides with mainly C26 acyl chains (Guillas et al.,
2001). However, this specificity can be modified by disrupting the
enzymes responsible for VLCFA synthesis, namely, ELO2 and
ELO3. Similarly, in the Arabidopsis pas3 mutant, elongation is
affected by the reduced cytoplasmic acetyl-CoA carboxylase
activity, and increased levels of C20 and C18 fatty acids are
found in sphingolipids (Roudier et al., 2010). These observations
indicate that there is a close relationship between the specificity
of the ceramide synthase reaction and the acyl-CoA pool feeding
that reaction; however, the nature of that relationship remains to
be understood.
Disruption of LOH1 and LOH3 also has a marked affect on the
total amount of sphingolipids. Free LCBs accumulate to high
levels and drive the synthesis of excessive amounts of 16:0containing sphingolipids. Similar findings were observed in the
CERS2 knockout mouse, which is depleted in VLCFA sphingolipids but has overall normal sphingolipid content due to the
accumulation of C16-sphingolipids (Pewzner-Jung et al., 2010).
Likewise, the reduction of the VLCFA-CoA pool in the elongation
mutants pas1 and pas3 led to an increase of C16-ceramides
(Roudier et al., 2010). Interestingly, sphingoid base hydroxylases
mutants that lacked t18:0 showed also enhanced C16-sphingolipids (Chen et al., 2008). In that article, a model was proposed
whereby disruption of t18:0 biosynthesis caused d18:0 to accumulate, which drove the synthesis of C16:0-containing sphingolipids through a C16:0-dependent ceramide synthase. It was also
suggested that t18:0-VLCFA–containing sphingolipids negatively
regulate serine palmitoyltransferase activity, regulating the amount
of sphingolipid that the cell synthesizes. This work further refines
that idea by identifying the C16:0-dependent ceramide synthase as
LOH2 and indicating that VLCFA-containing sphingolipids are
capable of affecting total sphingolipid content.
Our work demonstrated that the length of the ceramide acyl
chain has an important effect on the biological function of the
sphingolipid molecules. While loss of LOH2 function did not lead
to any obvious developmental phenotype, the loh1-2 loh3-2
double mutant was impaired in embryo development and germination, indicating that very-long-chain but not long-chain
ceramides are critical for plant growth. Fine-tuning of ceramide
synthase activity could be efficiently achieved with the sphingoid
base analog FB1. FB1 is a mycotoxin from Fusarium moniliforme
that inhibits ceramide synthase and is responsible for several
naturally and experimentally induced animal and human diseases (Desai et al., 2002; Stockmann-Juvala and Savolainen,
2008). FB1 was found to inhibit ceramide synthase and to lead
to free LCB accumulation (Abbas et al., 1994; Spassieva et al.,
2002). Sensitivity to FB1 was also described to directly correlate
with the activity of tomato LAG1 homolog Asc-1 (Brandwagt
et al., 2000, 2002; Spassieva et al., 2002).
Here, we demonstrated that FB1 selectively inhibits very-longacyl-chain ceramide synthesis in Arabidopsis. Moreover, our
results demonstrate that, at the concentrations used, the most of
the effects of FB1 can be attributed to the inhibition of de novo
ceramide synthesis since many FB1 phenotypes were observed
in the loh1 loh3 mutant. Interestingly, lateral roots were more
sensitive to FB1 than primary roots. Contrary to primary roots
that are of embryogenic origin, lateral roots are developed from
nondividing pericycle cells and exhibit extensive plasticity associated with differential response to environmental cues, like
phosphate or nitrate (Linkohr et al., 2002; Jain et al., 2007).
Lateral roots could thus be more sensitive to protein secretion at
the PM to respond to these different environmental stimuli.
The use of FB1 to inhibit ceramide synthesis in a short window
of time demonstrates the role of these lipids in the secretory
pathway in particular for two specific cargoes, PIN1-GFP and
AUX1-YFP. The normal expression and subcellular distribution
of AXR4-GFP in the presence of FB1 indicates that the effect of
sphingolipid depletion on AUX1-YFP targeting is independent of
the AXR4 pathway. Interestingly, PIN1, but not AUX1, targeting
was also impaired in VLCFA-defective mutants pas1 and 3
(Roudier et al., 2010). The fact that PIN1-GFP was more sensitive
to BFA in pas1 mutant suggests that PIN1 recycling was selectively altered. The differences observed between pas1 and FB1
or loh1 loh3 plants could be explained by the nature of the VLCFA
lipids involved since pas1 mutation impacts potentially all the
VLCFA-containing lipids and even among sphingolipids pas1
was found to reduce more the levels of glucosylceramides than
glucosylinositolphosphorylceramides (Roudier et al., 2010). PIN1
membrane localization was found to be dependent on sterol
levels, as illustrated by polarity defects in sterol methyltransferase mutant smt1orc (Willemsen et al., 2003). The role of sterols in
AUX1 polarity is debated since conflicting results were obtained
with smt1orc (Willemsen et al., 2003; Kleine-Vehn et al., 2006).
Nonetheless, the application of filipin, a specific sterol binding
drug, was able to specifically reduce sterol levels at the PM and
resulted in changes in AUX1 polar distribution and caused its
cytosolic aggregation similarly to what was found with FB1
(Kleine-Vehn et al., 2006).
Interestingly, PIN2 polarity was also dependent on sterol levels
since PIN2 localized to apical, basal, and lateral membranes in
the sterol biosynthetic mutant cpi-1 (Men et al., 2008). In contrast
with defects in sterol biosynthesis, inhibition of ceramide synthase did not seem to alter PIN2-GFP targeting, indicating a
potentially differential sensitivity of the membrane proteins to the
nature of their lipid environment. Similarly, we showed that FB1
treatment specifically altered the Rab-A2a– and Rab-A1e–labeled
early endosome populations. The fact that both markers are
targeted to the cell plate (Chow et al., 2008) indicates that VLCFA
sphingolipids might directly impact cytokinesis, as recently suggested in BY2 cells (Aubert et al., 2011). The existence of lipid
sorting at the TGN was recently demonstrated in yeast with
specific enrichment of sphingolipids and sterols in post-Golgi
vesicles transporting raft proteins (Klemm et al., 2009). Not
surprisingly, PIN1 was found to be associated with detergentresistant membrane fractions that are usually rich in sterols
and sphingolipids (Mongrand et al., 2004; Borner et al., 2005;
Titapiwatanakun et al., 2009). Detergent-resistant membrane,
enriched in sterol and sphingolipids, cofrationate with PM and
Golgi but not with ER proteins (Laloi et al., 2007). Inhibition of
Sphingolipids and Polar Auxin Transport
ceramide synthase did not seem to alter the ER and Golgi
structure even though Arabidopsis ceramide synthases were
found to localize in the ER (Marion et al., 2008). However, the
reduction of ceramide synthesis could impair the assembly of lipid
domains in the ER or Golgi, leading to subsequent disorganization
of specific endosomal populations.
Recently, FB1 was reported to induce ER-derived aggregates
and to block ER to Golgi cargo delivery in BY2 cells (Aubert et al.,
2011). The nature of the sphingolipids involved remain to be
found, but the acyl chain length was found to be critical for polar
targeting and trafficking and could not be compensated for by
shorter chain ceramides. Importantly, very-long-chain ceramides were a determinant of protein cargoes and endosomal
specificity. Very-long-chain fatty acyl chains were also described
to be involved in polar auxin transport in Arabidopsis (Roudier
et al., 2010). Reduction of the acyl chain length in particular in
sphingolipids resulted in PIN1-GFP but not AUX1-YFP cytosolic
aggregation. The relative insensitivity of AUX1-YFP to VLCFA
depletion in that report might be explained by a lesser reduction of very-long-acyl-chain sphingolipids compared with that
achieved in this work by FB1 treatment or the effect of the loh1-1
loh3-1 double mutant. However, enhanced sensitivity of PIN1
compared with AUX1 was also observed in this work, as PIN1
was aggregated in both weak and strong loh1 loh3 double
mutants, while AUX1 distribution was only impaired in the strong
loh1-2 loh3-2 mutant. These data suggest that AUX1 sensitivity
to lipid environment might be more complex than for PIN1.
The VLCFA content of lipids has been shown to affect membrane bending during yeast nuclear pore formation and membrane
plasticity during furrow ingression in Drosophila melanogaster
(Schneiter et al., 1996; Szafer-Glusman et al., 2008). Simulation of
lipid bilayers comprising common sphingomyelins showed that
increasing acyl chain length enhances bilayer thickness and lipid
packing but reduces lateral diffusion because of acyl chain interdigitations (Niemelä et al., 2006). Impairment of yeast fatty acid
elongation leading to reduced C26-sphingolipid levels decreased
lipid packing and membrane order in yeast cell but also prevented
liquid ordered phase separation in model membranes from total
lipid extracts (Klose et al., 2010). Such structural properties of the
acyl chains of sphingolipids could infer specific membrane features like vesicular mobility or fusions that could define some
endosomal populations and PM subdomains. However, the molecular mechanisms underlying these specificities remain to be
explored to fully understand the structural role of sphingolipids in
membrane dynamics.
METHODS
Bioinformatics and Phylogenetic Analysis
GenBank and the tomato genome database (http://mips.helmholtzmuenchen.de/plant/tomato/index.jsp) were searched using the tBLASTn
algorithm and the Asc-1 protein sequence AJ312131 using default parameters, except the filter for low complexity was turned off. The GenBank
sequence for Arabidopsis lyrata XM_002890624 was manually extended
using the A. lyrata sequence available at http://genome.jgi-psf.org/Araly1/
Araly1.home.html. Full-length eudicot sequences were identified and the
protein sequences aligned using ClustalW. Gaps were condensed and
edited using the BioEdit sequence software. Phylogenetic relationships
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between sequences were established using the MEGA4 software program
with parameters set to the default values (Tamura et al., 2007). Transmembrane domains were predicted with TMPred (Hofmann and Stoffel, 1993).
Plant Growth Condition and Drug Treatments
Seedlings were grown on Arabidopsis thaliana agar medium (Estelle and
Somerville, 1987) for phenotyping, genotyping, and drug treatment. FB1
(Sigma-Aldrich) short treatment was performed by incubating 9-d-old
seedlings in liquid Arabidopsis media for 16 to 20 h in the presence of 2.5
mM FB1. Due to batch variability, each FB1 stock solution was calibrated
with a root growth response curve and variation of drug concentration
never exceeded 2.5 6 1 mm for a short incubation. For long FB1
treatment, seedling were directly germinated on Arabidopsis solid media
supplemented with 0.25 to 3.5 mM FB1. BFA (Sigma-Aldrich), which
inhibits anterograde vesicular transport, was used in liquid Arabidopsis
media at 50 mM for 1 h on 9-d-old seedlings. After each drug treatment,
seedlings where washed before mounting for imaging.
Complementation of the lateral root defect of the loh1-1 loh3-1 mutant
or FB1-treated seedlings was obtained with 0.1 mM NAA (Sigma-Aldrich)
supplemented in solid Arabidopsis media with or without 0.5 mM FB1.
The different transgenic lines used in this study were as follows:
pPIN1:PIN1-GFP (Benková et al., 2003), pPIN2:PIN2-GFP (Abas et al.,
2006), pPIN2:PIN1-GFP (Wisniewska et al., 2006), pAUX1:AUX1-YFP
(Swarup et al., 2004), pLAX3:LAX3-GFP (Swarup et al., 2008), 35S:
PIP2:GFP and 35S:LTi6b:GFP (Cutler et al., 2000), pABC19:ABC19GFP and pABC1:ABC1-GFP (Titapiwatanakun et al., 2009), pSNX1:
SNX1-GFP (Jaillais et al., 2006), and pAXR4:AXR4-GFP (Dharmasiri
et al., 2006). The lines from the Wave Marker collection lines containing
the UBQ10 promoter were as follows: wave6 (YFP-NIP1), wave29Y
(YFP-ARA5/RabD2a), wave33Y (YFP-RAbD2b), wave18Y (YFP-GOT1),
wave127Y (YFP-MEMB), wave22Y (YFP-SYP32), wave34Y (YFPRABA1e), wave2Y (YFP-ARA7), wave5Y (YFP-RABG3F), wave9Y (YFPVAMP711), and wave11Y (YFP-RabG3C) (Geldner et al., 2009). The
Rab-A2a mutant constructs were the following: pRab-A2a:YFP-pRabA2a, pRab-A2a:YFP-Rab-A2a[N125I], pRab-A2a:YFP-Rab-A2a[S26N],
pRab-A2a:YFP-Rab-A2a[Q71L] (Chow et al., 2008), pBOR1-BOR1GFP (Takano et al., 2005), and p35S:LeAGP1-GFP (Estévez et al.,
2006).
Insertion Mutants
Two Arabidopsis ecotypes Columbia-0 (Col-0) and Wassilewskija (Ws)
were used. loh1-1, loh2-1, and loh3-1 are in the Ws background and were
isolated by screening the original collection of 60,480 T-DNA insertions
described by Krysan et al. (1999) using the following primers: loh1-1
(59-TCTTTTATCTCATTCTCCTTTGCTCTGCT-39; 59-GAACCAAAAAAAACCCCAAGAAAATTCAT-39), loh2-1 (59-AGTCAGTAAGGTAATGAAGCTGAAAACAA-39; 59-AAAAAGAAAGAAGGTCTGTGAGATCCCAA-39),
and loh3-1 (59-ATTCGATTTCTCCTCGATAGATTCGTCTT-39; 59-ACAACGTGCTTTTACATTTCTATCTACCT-39) in combination with the left
border primer JL202 (59-CATTTTATAATAACGCTGCGGACATCTAC-39).
loh1-2 (N580371), loh2-2 (N524192), and loh3-2 (N650849) were obtained
from the Salk collection. The genotyping of SALK loh was performed
with the following primers: loh1 (59-TTCCTTCTTATGTGATTGTAAAGAGAA-39; 59-AAAAACCCCAAGAAAATTCATTTAG-39), loh2 (59-TTTCGGATTCTTCTTCTTGA-39, 59-AAAGACATCACTTGCATCATG-39), and loh3
(59-TGGTTTAAAGAGAGGATTTTACGG-39, 59-CAAAATTTGTTCAGACTACAATTCAA-39).
Lipid Analysis
Free and total LCB analysis was performed by HPLC after fluorescent
derivatization (Bach et al., 2008). Complete sphingolipid analysis was
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performed by mass spectrometry as described (Markham and Jaworski,
2007).
Auxin Transport Measurements
Auxin transport was measured through sections cut from 12- to 15-cmlong inflorescence bolts as previously described (Brown et al., 2001). Ws
and loh1-1 loh3-1 plants were grown for 5 to 6 weeks until the bolts were
12 to 15 cm high. Sections 25 mm in length were cut using a sharp razor
blade from the bottom, the middle, and 10 mm below the top of the bolt.
The stem section was immediately placed in a tube containing 20 mL of 5
mM MES, pH 5.5, 1% Suc, 0.05% Tween 20, 19 nCi 1-14C-indole acetic
acid (9 mCi/mmol) with or without the addition of 10 mM NPA such that
one end of the stem segment was immersed in the solution. To measure
basipetal transport, the stem segment was placed in the inverted orientation, that is, with the top of the stem segment in the solution. Stem
segments were incubated for 16 h in the dark after which the upper 5 mm
of the stem was excised, immersed in scintillation fluid, and the dpm
measured in a scintillation counter.
Live-Cell Imaging
Excitation/emission of YFP (514 nm, 520 to 560 nm) GFP (488 nm, 490 to
550 nm), and FM4-64 (488 or 514 nm, 600 to700 nm) was performed on
LSM 710 (Zeiss) and SP2 (Leica) confocal microscopes. Root tip imaging
was performed with seedlings mounted in water, and high magnification
images were taken with a 363 objective lens (water immersion) with a line
averaging of 8. Roots were stained with 2 mM FM4-64 and directly
mounted on the microscope slide. Spectral separation of PIN1:GFP and
AUX1:YFP was conducted according the GFP and YFP emission profile
and the LSM software spectral mode. GUS and propidium iodide staining
was performed as described previously (Harrar et al., 2003; Truernit et al.,
2008). FRAP analysis of PIN1-GFP and AUX1-YFP was performed on 10to 12-d-old secondary root tips of pPIN1:PIN1-GFP and pAUX1:AUX1YFP seedlings treated with 2.5 mM FB1. The FRAP experiment was
performed using a Zeiss LSM710 with a 340 oil objective. A prebleach
scan was done with either a 488-nm laser excitation (5%) for PIN1-GFP or
a 514-nm laser excitation (3.5%) for AUX1-YFP with a pinhole of 82.2 and
a 512 3 512 (8-bit) acquisition mode with a pixel dwell of 1.27 ms, 8 line
average, and a 23 zoom. Fluorescence bleaching was performed after
three prebleach scans with a double scan at 100% laser 488 and 514 nm
and a pixel dwell of 177.32 ms. Confocal pictures were processed using
ImageJ V1.44m. The pictures corresponding to prebleach, postbleach,
and each recovery time point (15, 30, 45, and 60 min) were assembled into
a time-lapse picture stack. Occasional drifting of the sample was corrected
using the StackReg plugin. About two to four cells were quantified per root.
Fluorescence intensity was measured on cell apical/basal membranes either
as rectangular region of interest (ROI) in the case of PMs or as circular ROI in
the case of FB1 aggregates. A nonbleached PM ROI was measured and
used to correct fluorescence decrease due to photobleaching during
acquisition. Fluorescence recovery was expressed as percentage of fluorescence increase of the bleached region corrected by the acquisition
bleaching factor of the whole sample.
8 h, before the next warm-up step to 2308C over 15 h, where they remain
for additional 8 h. Root tips were finally infiltrated and embedded in epoxy
resin (low viscosity Premix Kit medium, agar) at room temperature
according to the manufacturer’s instructions. For polymerization, they
were placed in flat plastic molds and polymerized for 17 h at 608C.
Electron Microscopy Observations
The 70-nm ultrathin sections (Ultracut UC6; Leica) were collected on
formvar-coated copper grids and poststained with aqueous 2% uranyl
acetate/lead citrate as described by Hawes and Satiat-Jeunemaitre
(2001). They were examined with a JEOL 1400 transmission electron
microscope operating at 120 kV. Images were acquired using a postcolumn high-resolution (11 megapixels) high-speed camera (SC1000
Orius; Gatan).
Experimental Procedures for Supplemental Data
The mRNA levels of LOH1, 2, and 3 were checked via RT-PCR for loh1-2,
loh2-2, and loh3-2 and via RNA gel blot on total RNA for loh1-1, loh2-1,
and loh3-1 using an RNA probe constructed from the cloned cDNA for
each gene (see Supplemental Figures 2A and 2B online).
Myriocin treatment was performed for 16 to 20 h on 9-d-old seedlings
at 7 mM directly dissolved in liquid Arabidopsis media.
The FDA test was performed on 9-d-old Col-0 or AUX1:YFP seedlings
treated with FB1 (16-h treatment at 2.5 mM). Seedlings were treated with
2 mM FDA for 15 min and then washed and directly mounted in 2 mM FM
4-64 for visualization (see Supplemental Figure 6B online). Esterase
activity of cells was monitored in the green and FM4-64 in red channel.
Both were activated with a 488-nm argon laser.
To test the effect of FB1 treatment on endocytosis, BOR11-GFP
seedlings grown for 5 d on agar medium were treated or not with 2.5 mM
FB1 (16 h) in water and mounted (t = 0) in 100 mM boric acid (62.5 mM
FB1) (see Supplemental Figure 10B online). To test the effect of FB1
treatment on secretion, 12-d-old seedlings (35S:LeAGP1-GFP) treated or
not with 2.5 mM FB1 (16 h) were plasmolysed using 0.4 mM mannitol with
3 mM propidium Iodide during 5 min and then mounted (see Supplemental
Figure 11F online).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
database or the Arabidopsis Genome Initiative database under the
following accession numbers: Arabidopsis (At) LOH1 (At3g25540),
LOH2 (At3g19260), LOH3 (At1g13580), and LOH4 (At1g26200). Accession numbers of tomato (Le) Asc1, Asc2, and Asc3 and yeast Lag1p and
Lac1p used in Figure 1A are AJ312131, SL1.00sc02793_15.1.1,
SL1.00sc02749_302.1.1, SCU08133, and NM_001179574, respectively.
The accession numbers of the Arabidopsis mutants used in this study are
as follows: loh1-2 (N580371), loh2-2 (N524192), loh3-2 (N650849), loh1-1
(CS66113), loh2-1 (CS66114), and loh3-1 (CS66115).
Supplemental Data
High-Pressure Freezing, Freeze Substitution, and
Embedding Processes
For electron microscopy, root tips (3 mm) were cut in 1-hexadecene,
transferred to 200-mm size cupules (Leica) containing 1-hexadecen, and
frozen with a high-pressure freezer apparatus (EMPACT2; Leica). Freeze
substitution was performed (Leica freeze substitution unit AFS2) in
acetone supplemented with 2% osmium tetroxide warming up progressively from 290 to 2308C (specimens are left at 2908C for 27 h, then
warmed up to 2608C over 15 h). Specimens stayed in a 2608C bath for
The following materials are available in the online version of this article.
Supplemental Figure 1. Protein Sequence Alignment of 29 LAG1
Homologs and Phylogenetic Relationship among LAG1 Homologs.
Supplemental Figure 2. Characterization of loh Mutants.
Supplemental Figure 3. Complementation of the loh1 loh3 Double
Mutant.
Supplemental Figure 4. Fatty Acid Content of Total Sphingolipids
and Free LCBs from loh Mutants.
Sphingolipids and Polar Auxin Transport
Supplemental Figure 5. Sphingolipid Analysis of FB1-Treated Seedlings.
Supplemental Figure 6. Effect of FB1 on Root Growth and Viability.
Supplemental Figure 7. FB1 Has No Effect on Several Membrane
Markers.
Supplemental Figure 8. Subcellular Distribution of PIN1-GFP and
AUX1-YFP and YFP-VAMP711 upon Sphingolipid Depletion.
Supplemental Figure 9. Endomembrane Ultrastructure Is Altered in
loh1-1/loh3-1 Root Cells.
Supplemental Figure 10. Involvement of Endocytosis in YFP-RabA2a
Aggregation.
Supplemental Figure 11. Involvement of Endocytic and Secretory
Pathways in YFP-RabA2a Aggregation.
Supplemental Data Set 1. Text File of the Sequences and Alignment
Used for the Phylogenetic Analysis Shown in Supplemental Figure 1B.
ACKNOWLEDGMENTS
D.M. and this work were supported by the Agence Nationale de la
Recherche Programe Blanc Sphingopolar (07-BLAN-202) and the European training program Marie Curie (European Union Versailles-Evry
Research Training program). J.E.M. was supported by National Science
Foundation Grant MCB 0312559 under the guidance of J.G. Jaworski
and by the University of Groningen in the laboratory of J. Hille. We thank
Thierry Gaude, Jan Traas, Markus Geisler, Christian Luschnig, and Jiřı́
Friml for the gifts of p35S:YFP-RAB-F2b, pPIN1-PIN1:GFP, pABCs:
ABCs-GFP, pPIN2:PIN2-GFP, and pPIN2:PIN1-GFP transgenic lines,
respectively. We thank Niko Geldner for all the wave lines. We thank
Ranjan Swarup for the gift of pAXR4:AXR4-GFP and pLAX3:LAX3-GFP
and for helpful discussion. We are grateful to Ian Moore for providing the
different pRab-A2:YFP-Rab-A2a mutant lines, to José Estevez for the
LeAGP1-GFP line, and to Junpei Takano for the BOR1-GFP line. We
thank Bruno Letarnec for taking care of the plants. We used the cytology
and imaging facility of the Plateforme de Cytologie et Imagerie Végétale
and the Plateforme de Chimie du Végétal of Institut Jean-Pierre Bourgin
(supported by Région Ile de France and Conseil Général des Yvelines)
and the electron microscopy facilities and cell biology unit of the Imagif
platform (Centre National de la Recherche Scientifique), supported by
the Conseil Général de l’Essonne.
Received October 13, 2010; revised April 15, 2011; accepted May 20,
2011; published June 10, 2011.
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Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific
Polar Plasma Membrane Protein Targeting in Arabidopsis
Jonathan E. Markham, Diana Molino, Lionel Gissot, Yannick Bellec, Kian Hématy, Jessica Marion,
Katia Belcram, Jean-Christophe Palauqui, Béatrice Satiat-JeuneMaître and Jean-Denis Faure
Plant Cell; originally published online June 10, 2011;
DOI 10.1105/tpc.110.080473
This information is current as of June 18, 2017
Supplemental Data
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